Bacteria undergo specific cell shape changes and localize various proteins to subcellular sites, both of which are important for their survival in te environment. For example, filamentation of uropathogenic E. coli subverts innate defenses during the infection process, and the helical shape of Helicobacter pylori is important for colonization of the stomach. Understanding how bacteria achieve changes in morphology and modes of growth and couple them to the localization of proteins, such as virulence factors, is critical for our ability to inhibit their persistence, proliferation, and host infection. The generl goal of this research is to study the mechanisms that generate various cell shapes and growth patterns and how they are coordinated with protein localization and function. This study takes advantage of stalk synthesis, a specialized zonal mode of growth in Caulobacter crescentus and its relatives that generates thin extensions of the cell envelope and of the recently discovered reproduction of bacteria in the Rhizobiales by polar growth, including some human pathogens. The project has three major aims. The first aim is to determine the role of the penicillin binding protein PbpC in the recruitment of the StpX protein to the stalk. This will be achieved by determining if StpX is inserted into the stalk concurrently with stalk elongation and if PbpC recruits StpX to the stalk by protein-protein interaction or through its enzymatic activity and modification of stalk peptidoglycan. A novel high throughput microscopy screen will be used to identify genes involved in protein targeting to the stalk and in stalk synthesis. The second aim is to determine how the muramidase SpmX is required for stalk synthesis in Asticcacaulis biprosthecum and directs its subcellular location. This will be accomplished by studying the muramidase activity of SpmX and determining its requirement for stalk synthesis and localization, for its own localization, and for the localization of the developmental regulator Div. Protein chimeras will be constructed to determine how the domains of SpmX have evolved to generate species-specific elements of each function. The effect of altering the localization of developmental regulators on developmental outcomes will also be studied. The third aim is to determine how bacteria in the Rhizobiales, including some human pathogens, reproduce by zonal growth at their pole. This will be accomplished by using Agrobacterium tumefaciens as a model to determine the growth mechanism in this group. Genes required to direct polar growth will be identified by candidate and high throughput approaches. Peptidoglycan composition will be analyzed in wild-type and specific mutants to determine if polar growth requires peptidoglycan synthesis and composition that are different than for lateral cell wall synthesis. Finally, the mechanisms by which peptidoglycan synthesis is redirected during the cell cycle will be determined. Insights gained from these studies can be used to design strategies to inhibit growth, prevent key morphological changes, or alter important protein localization pathways in pathogens, thereby improving our ability to control them. PUBLIC HEALTH RELEVANCE: Bacteria undergo specific cell shape changes and localize various proteins to subcellular sites, both of which are important for their survival in the environment, including the host environment for pathogen. We will study the mechanisms that generate various cell shapes and growth patterns and how they are coordinated with and influence protein localization and function. Insights gained from these studies can be used to design strategies to inhibit growth, prevent key morphological changes, or alter important protein localization pathways in pathogens, thereby improving our ability to control them.